Construction method of characteristic chromatogram of traditional Chinese medicine compound preparation for treating chronic pelvic inflammation and application thereof

Abstract

The application relates to the technical field of traditional Chinese medicine detection, in particular to a construction method of a characteristic chromatogram of a traditional Chinese medicine compound preparation and application thereof. The traditional Chinese medicine compound preparation is Taohong Siwu decoction or a freeze-dried agent thereof or other preparations prepared from pharmaceutically acceptable adjuvants; the construction method of the characteristic chromatogram comprises the following steps: dissolving the traditional Chinese medicine compound preparation in a solvent to prepare a test sample solution; performing liquid chromatography detection on the test sample solution to prepare a characteristic chromatogram of the test sample; and the liquid chromatography detection conditions comprise: using methanol as mobile phase A, using acetonitrile as mobile phase B, and using 0.1%-0.3% phosphoric acid aqueous solution as mobile phase C. The characteristic chromatogram constructed by the application has a smooth baseline, suitable peak height, good peak shape, small interference peak influence, 16 common peaks are obtained through detection, 12 index components are identified, and the content determination is accurate, and the result is reliable, scientific and objective.

Description

Technical Field

[0001] This application relates to the field of traditional Chinese medicine detection technology, and in particular to a method for constructing a characteristic spectrum of a traditional Chinese medicine compound preparation for treating chronic pelvic inflammatory disease and its application, wherein the traditional Chinese medicine compound preparation for treating chronic pelvic inflammatory disease is a Taohong Siwu Decoction preparation. Background Technology

[0002] The traditional Chinese medicine compound Taohong Siwu Decoction is derived from Siwu Decoction. Its medicinal composition was first recorded in the Ming Dynasty book "Yuji Weiyi", and its name was first recorded in "Yizong Jinjian". The famous gynecological work "Fuke Bingjian: Menstruation Section: Menstrual Delay" written by Chai Dehua in the Qing Dynasty first clearly recorded the ingredients, processing methods, and dosage of Taohong Siwu Decoction. In 2018, the State Administration of Traditional Chinese Medicine and the National Medical Products Administration jointly issued the "Catalogue of Ancient Classic Prescriptions (First Batch)" (the "Catalogue"), which included Taohong Siwu Decoction and recognized it as a classic prescription for treating menstrual disorders and blood deficiency with blood stasis. This formula consists of Angelica sinensis, Rehmannia glutinosa, Ligusticum chuanxiong, Paeonia lactiflora, Prunus persica, and Carthamus tinctorius. Rehmannia glutinosa and Angelica sinensis nourish and invigorate blood, serving as the principal herbs. Ligusticum chuanxiong invigorates blood and removes stagnation, Paeonia lactiflora nourishes blood and astringes yin, while Carthamus tinctorius and Prunus persica break up blood stasis, promote blood circulation, and generate new blood. Together, they serve as the assistant herbs, possessing the effects of nourishing blood, invigorating blood, regulating menstruation, and relieving pain. Modern medicine has confirmed its significant efficacy in treating chronic pelvic inflammatory disease. Traditional Chinese medicine compound preparations are composed of multiple herbs, exerting their therapeutic effects through the synergistic action of multiple components and targets. Therefore, studying their chemical composition is of great significance. Thus, qualitative and quantitative research on the material basis is crucial when developing this formula.

[0003] Characteristic chromatograms of traditional Chinese medicine (TCM) can rapidly identify common peaks in various components of TCM compound preparations, serving as a comprehensive and quantifiable identification method commonly used to study the uniformity and stability of TCM quality. Furthermore, multi-index component content determination methods based on common peaks in characteristic chromatograms can provide comprehensive quality control evaluation of TCM compound preparations at both qualitative and quantitative levels. Currently, the characteristic chromatograms and / or fingerprint chromatograms of Taohong Siwu Decoction cannot fully reflect the material basis of its medicinal materials, necessitating the development of new characteristic chromatograms for Taohong Siwu Decoction. Summary of the Invention

[0004] Based on this, the purpose of this application includes providing a method for constructing a characteristic spectrum of a traditional Chinese medicine compound preparation for treating chronic pelvic inflammatory disease and its application, which can more fully characterize the medicinal information in the Taohong Siwu Decoction preparation and has better specificity and comprehensiveness.

[0005] The first aspect of this application provides a method for constructing a characteristic spectrum of a traditional Chinese medicine compound preparation for treating chronic pelvic inflammatory disease, characterized in that the traditional Chinese medicine compound preparation for treating chronic pelvic inflammatory disease is a Taohong Siwu Decoction preparation.

[0006] The method for constructing the feature map includes the following steps:

[0007] The traditional Chinese medicine compound preparation was dissolved in a solvent to prepare a test solution;

[0008] The test solution was subjected to liquid chromatography to obtain a characteristic chromatogram of the test sample.

[0009] The conditions for the liquid chromatography detection include:

[0010] (1) Methanol is used as mobile phase A, acetonitrile is used as mobile phase B, and 0.1%~0.3% phosphoric acid aqueous solution is used as mobile phase C;

[0011] (2) Gradient elution is employed, wherein the gradient elution procedure includes:

[0012] From 0 to 5 minutes, maintain the volume percentage of mobile phase A at 5%, the volume percentage of mobile phase B at 0%, and the volume percentage of mobile phase C at 95%.

[0013] From 5 min to 15 min, the volume percentage of mobile phase A increases from 5% to 15%, the volume percentage of mobile phase B is maintained at 0%, and the volume percentage of mobile phase C decreases from 95% to 85%.

[0014] Between 15 and 18 minutes, the volume percentage of mobile phase A decreased from 15% to 0%, the volume percentage of mobile phase B increased from 0% to 10%, and the volume percentage of mobile phase C increased from 85% to 90%.

[0015] From 18 min to 45 min, the volume percentage of mobile phase A is maintained at 0%, the volume percentage of mobile phase B increases from 10% to 20%, and the volume percentage of mobile phase C decreases from 90% to 80%.

[0016] For 45-55 minutes, the volume percentage of mobile phase A is maintained at 0%, the volume percentage of mobile phase B increases from 20% to 40%, and the volume percentage of mobile phase C decreases from 80% to 60%.

[0017] For 55-60 minutes, the volume percentage of mobile phase A is maintained at 0%, the volume percentage of mobile phase B increases from 40% to 85%, and the volume percentage of mobile phase C decreases from 60% to 15%.

[0018] For 60-70 minutes, the volume percentage of mobile phase A is maintained at 0%, the volume percentage of mobile phase B increases from 85% to 95%, and the volume percentage of mobile phase C decreases from 15% to 5%.

[0019] A second aspect of this application provides the application of a method for constructing a characteristic spectrum of the traditional Chinese medicine compound preparation for treating chronic pelvic inflammatory disease, for use in at least one of the following (1) to (3):

[0020] (1) Identify the authenticity of the traditional Chinese medicine compound preparation for treating chronic pelvic inflammatory disease;

[0021] (2) To evaluate the quality of the traditional Chinese medicine compound preparation for treating chronic pelvic inflammatory disease;

[0022] (3) The content of each indicator component in the traditional Chinese medicine compound preparation for treating chronic pelvic inflammatory disease was determined.

[0023] This application establishes a method for constructing a characteristic spectrum of a traditional Chinese medicine compound preparation for treating chronic pelvic inflammatory disease by establishing suitable high-performance liquid chromatography conditions. The obtained characteristic spectrum can more comprehensively reflect the intrinsic chemical information of the traditional Chinese medicine compound and provide a reference for the research of the material basis of the traditional Chinese medicine compound.

[0024] The characteristic chromatogram of the traditional Chinese medicine compound preparation for treating chronic pelvic inflammatory disease in this application includes at least 16 common peaks, reflecting rich intrinsic chemical information, and identifies at least 12 components. These components can serve as indicator components of the traditional Chinese medicine compound preparation. By detecting the presence and / or content of each indicator component in the test substance, the authenticity and quality of the test substance can be identified. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0026] Figure 1 This is a comparison graph of the liquid phase detection of the test solution and the reference solution in Example 1 of this application; Figure 1 A in the figure is the liquid chromatogram of the compound preparation standard used for reference; Figure 1 B is a superimposed image of the liquid chromatograms of 15 batches of test solution (S1-S15); Figure 1 The labels and corresponding peaks appearing in the sample are as follows: 1. Gallic acid; 3. Rehmannia glycoside D; 5. D-amygdalin; 6. Chlorogenic acid; 7. Hydroxysafflower yellow pigment A; 8. Paeoniflorin; 9. Paeoniflorin; 10. Ferulic acid; 11. Verbascoside; 13. Ligusticum lactone I; 14. Ligusticum lactone H; 15. Ligusticum lactone A; 16. Ligusticum lactone;

[0027] Figure 2This is a comparison graph of the liquid chromatography detection of the mixed reference standard and the test solution in Example 1 of this application; Figure 2 A is the liquid chromatogram of the reference solution; Figure 2 B in the figure is the liquid chromatogram of the analyte solution; Figure 2 The labels and corresponding peaks appearing in the sample are as follows: 1. Gallic acid; 2. Rehmannia glutinosa D; 3. Amygdalin; 4. Chlorogenic acid; 5. Hydroxysafflower yellow A; 6. Paeoniflorin; 7. Paeoniflorin; 8. Ferulic acid; 9. Verbascoside; 10. Ligusticum striatum lactone I; 11. Ligusticum striatum lactone H; 12. Ligusticum striatum lactone A; 13. Ligusticum striatum lactone;

[0028] Figure 3 The chromatograms of the test solution subjected to liquid chromatography detection under different chromatographic conditions in Comparative Example 1 of this application are shown.

[0029] Figure 4 The chromatograms are for comparative example 2 of this application, obtained by liquid chromatography detection of the test solution under different chromatographic conditions. Detailed Implementation

[0030] The present application is further described below with reference to embodiments and examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Furthermore, it should be understood that after reading the teachings of this application, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also fall within the protection scope of the appended claims.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0032] the term:

[0033] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0034] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations encompass any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that, in this application, the technical solution undoubtedly includes solutions connected by "logical AND," and also undoubtedly includes solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0035] In this document, terms such as "preferred," "better," and "more preferred" are merely descriptions of implementation methods or examples that achieve better results, and should be understood as not constituting a limitation on the scope of protection of this application.

[0036] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0037] In this application, the terms "first aspect," "second aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first aspect," "second aspect," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0038] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0039] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, optional numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0040] In this application, weight can be a well-known unit of mass in the chemical industry, such as μg, mg, g, or kg.

[0041] In one aspect of this application, a method for constructing a characteristic spectrum of a traditional Chinese medicine compound preparation for treating chronic pelvic inflammatory disease is provided, wherein the traditional Chinese medicine compound preparation for treating chronic pelvic inflammatory disease is a Taohong Siwu Decoction preparation;

[0042] The method for constructing its feature map includes the following steps:

[0043] The tested Taohong Siwu Decoction preparation was dissolved in a solvent to obtain the test solution.

[0044] The test solution was subjected to liquid chromatography to obtain a characteristic chromatogram of the test sample.

[0045] The conditions for liquid chromatography detection include:

[0046] (1) Methanol is used as mobile phase A, acetonitrile is used as mobile phase B, and 0.1%~0.3% phosphoric acid aqueous solution is used as mobile phase C;

[0047] (2) Gradient elution is used. The gradient elution procedure includes:

[0048] From 0 to 5 minutes, maintain the volume percentage of mobile phase A at 5%, the volume percentage of mobile phase B at 0%, and the volume percentage of mobile phase C at 95%.

[0049] From 5 min to 15 min, the volume percentage of mobile phase A increased from 5% to 15%, while the volume percentage of mobile phase B remained at 0%, and the volume percentage of mobile phase C decreased from 95% to 85%.

[0050] Between 15 and 18 minutes, the volume percentage of mobile phase A decreased from 15% to 0%, the volume percentage of mobile phase B increased from 0% to 10%, and the volume percentage of mobile phase C increased from 85% to 90%.

[0051] From 18 min to 45 min, the volume percentage of mobile phase A was maintained at 0%, the volume percentage of mobile phase B increased from 10% to 20%, and the volume percentage of mobile phase C decreased from 90% to 80%.

[0052] From 45 min to 55 min, the volume percentage of mobile phase A was maintained at 0%, the volume percentage of mobile phase B increased from 20% to 40%, and the volume percentage of mobile phase C decreased from 80% to 60%.

[0053] From 55 to 60 minutes, the volume percentage of mobile phase A was maintained at 0%, the volume percentage of mobile phase B increased from 40% to 85%, and the volume percentage of mobile phase C decreased from 60% to 15%.

[0054] For 60-70 minutes, the volume percentage of mobile phase A was maintained at 0%, the volume percentage of mobile phase B increased from 85% to 95%, and the volume percentage of mobile phase C decreased from 15% to 5%.

[0055] In one embodiment, the wavelengths for detection by liquid chromatography include:

[0056] The detection wavelength is 275nm~285nm from 0min to 16min.

[0057] 16 min to 30 min, detection wavelength 205 nm to 215 nm;

[0058] 30 min to 37 min, detection wavelength 225 nm to 235 nm;

[0059] 37 min to 48 min, detection wavelength 325 nm to 335 nm;

[0060] The detection wavelength was 275nm to 285nm, lasting from 48 to 51 minutes.

[0061] 51 min to 54 min, detection wavelength 400 nm to 405 nm;

[0062] The detection time is 54 min to 70 min, and the detection wavelength is 275 nm to 285 nm.

[0063] In one embodiment, the conditions for liquid chromatography detection further include at least one of the following:

[0064] (1) The flow rate is 0.8 mL / min to 1.2 mL / min;

[0065] (2) The injection volume is 4 μL to 6 μL;

[0066] (3) The column temperature is 28℃~32℃;

[0067] (4) The chromatographic column is a C18 column.

[0068] In one embodiment, the method for constructing the characteristic spectrum of a traditional Chinese medicine compound preparation for treating chronic pelvic inflammatory disease further includes the following steps:

[0069] A reference solution is provided, which includes gallic acid and a solvent;

[0070] The reference solution was subjected to liquid chromatography to obtain a characteristic spectrum of the reference.

[0071] By comparing the characteristic spectrum of the reference material with the characteristic spectrum of the test sample, a characteristic spectrum of the traditional Chinese medicine compound preparation for treating chronic pelvic inflammatory disease was prepared.

[0072] The conditions for liquid chromatography detection are as described in any of the above technical solutions for liquid chromatography detection of the test sample, and will not be repeated here.

[0073] In one embodiment, the preparation steps of the traditional Chinese medicine compound preparation include: taking wine-processed Rehmannia glutinosa, wine-processed Angelica sinensis, wine-processed Paeonia lactiflora, Ligusticum chuanxiong, Prunus persica and wine-processed Carthamus tinctorius, adding 11 to 13 times the amount of water, bringing it to a boil over high heat, and then simmering it over low heat for 20 to 30 minutes.

[0074] Preferably, the mass ratio of wine-processed Rehmannia glutinosa, wine-processed Angelica sinensis, wine-processed Paeonia lactiflora, Ligusticum chuanxiong, Prunus persica and wine-processed Carthamus tinctorius is (55~57):(74~76):(27~29):(18~20):(19~21):(18~20).

[0075] In one embodiment, the characteristic spectrum prepared by the method for constructing the characteristic spectrum of the traditional Chinese medicine compound preparation includes 16 characteristic peaks;

[0076] Among them, peak 1 (gallic acid) is the reference peak, and the remaining characteristic peaks and their relative retention times are as follows: peak 2: 1.08±5%; peak 3: 1.27±5%; peak 4: 1.85±5%; peak 5: 2.14±5%; peak 6: 2.23±5%; peak 7: 2.55±5%; peak 8: 2.64±5%; peak 9: 2.88±5%; peak 10: 3.47±5%; peak 11: 3.78±5%; peak 12: 4.10±5%; peak 13: 4.13±5%; peak 14: 4.23±5%; peak 15: 4.73±5%; peak 16: 4.81±5%.

[0077] Furthermore, among the 16 characteristic peaks:

[0078] Peak 1 is gallic acid, peak 3 is rehmannia glycoside D, peak 5 is D-amygdalin, peak 6 is chlorogenic acid, peak 7 is hydroxysafflower yellow pigment A, peak 8 is paeoniflorin, peak 9 is paeoniflorin, peak 10 is ferulic acid, peak 11 is verbascoside, peak 13 is ligustilide I, peak 14 is ligustilide H, peak 15 is ligustilide A, and peak 16 is ligustilide.

[0079] In a second aspect of this application, the application of a method for constructing a characteristic spectrum of a traditional Chinese medicine compound preparation for treating chronic pelvic inflammatory disease as described in any of the above technical solutions is provided, for use in at least one of the following (1) to (3):

[0080] (1) Identify the authenticity of traditional Chinese medicine compound preparations for the treatment of chronic pelvic inflammatory disease;

[0081] (2) To evaluate the quality of traditional Chinese medicine compound preparations for the treatment of chronic pelvic inflammatory disease;

[0082] (3) The content of indicator components in traditional Chinese medicine compound preparations for the treatment of chronic pelvic inflammatory disease was determined.

[0083] Preferably, the indicator components in the traditional Chinese medicine compound preparation for treating chronic pelvic inflammatory disease include gallic acid, rehmannia glycoside D, amygdalin, chlorogenic acid, hydroxysaffron yellow A, paeoniflorin, paeoniflorin, ferulic acid, verbascoside, ligustrolide I, ligustrolide H and ligustrolide A.

[0084] In one embodiment, the content and determination of various indicator components in a traditional Chinese medicine compound preparation for treating chronic pelvic inflammatory disease include:

[0085] The traditional Chinese medicine compound preparation to be tested is dissolved in a solvent to prepare a solution of the test sample;

[0086] A reference solution is provided, which includes the indicator component and the solvent;

[0087] The test solution and the reference solution were subjected to liquid chromatography for detection, and the content of each indicator component in the test solution was calculated.

[0088] The conditions for liquid chromatography detection are as described in any of the above technical solutions for liquid chromatography detection of the test sample, and will not be repeated here.

[0089] The following are some specific examples.

[0090] For experimental parameters not specified in the following specific embodiments, please refer to the guidelines given in this application document first, or refer to experimental manuals or other experimental methods known in the art, or refer to the experimental conditions recommended by the manufacturer.

[0091] The raw materials and reagents involved in the following specific embodiments can be obtained commercially or prepared by those skilled in the art using known methods.

[0092] Example 1

[0093] In this embodiment, suitable chromatographic conditions were established, and the Taohong Siwu Decoction preparation was detected. Based on the detection by liquid chromatography, its characteristic chromatogram was established, and the methodology was validated.

[0094] 1. Materials

[0095] 1.1 Instruments

[0096] Agilent 1260 HPLC system (including G7111 vacuum degasser, G711B quaternary pump, G7129A autosampler, G7116A column oven, and G7117C DAD detector, Agilent Technologies, USA); BT-25S, BT-124S, and BT-323S analytical balances (Sartorius Scientific Instruments (Beijing) Co., Ltd.); ZNHW electric heating mantle (Shanghai Qiangqiang Instrument Equipment Co., Ltd.); HWS-26 electric thermostatic water bath (Shanghai Yiheng Scientific Instruments Co., Ltd.); 45u2 purple clay decoction pot (Chaozhou City, Chaoan District, Kangyashun Electric Appliance Co., Ltd.); BDZ-155-B portable gas stove (Zhejiang Yongkang City, Jinyu Co., Ltd.).

[0097] 1.2 Drug Testing

[0098] Gallic acid (batch number: 110831-201906), amygdalin (batch number: 110820-201808), hydroxysaffron yellow A (batch number: 111637-201810), verbascoside (batch number: 111530-201713), chlorogenic acid (batch number: 110753-201817), paeoniflorin (batch number: 110736-201943), and ferulic acid (batch number: 110773-201614) were all purchased from the National Institutes for Food and Drug Control. Rehmannia glutinosa D reference standard (batch number: D-084-200103, purchased from Chengdu Ruifensi Biotechnology Co., Ltd.); paeoniflorin reference standard (batch number: DST190912-071, purchased from Chengdu Lemeitian Pharmaceutical Technology Co., Ltd.); ligustrol I (batch number: AF9102004), ligustrol H (batch number: AF9102005), ligustrol A (batch number: AF9102006), and ligustilide (batch number: AF9102001) reference standards were all purchased from Chengdu Efa Biotechnology Co., Ltd.; the HPLC purity of the above reference standards was ≥98% as determined by the area normalization method. Acetonitrile, methanol, and phosphoric acid were of chromatographic grade (Fisher Laboratories, USA); other reagents were of analytical grade.

[0099] Safflower, peach kernel, white peony root, rehmannia root, angelica root, and chuanxiong rhizome were purchased from their respective authentic producing areas and main production areas. They were identified by researcher Wu Lihong of the Shanghai Center for Standardization of Traditional Chinese Medicine. The identification results showed that: safflower is the dried flower of *C. tinctorius* L. (Asteraceae); peach kernel is the dried mature seed of *P. davidiana* (Carr.) Franch. (Rosaceae); white peony root is the dried root of *P. lactiflora* Pall. (Ranunculaceae); rehmannia root is the dried tuberous root of *R. glutinosa* Libosch. (Scrophulariaceae); angelica root is the dried root of *A. sinensis* (Oliv.) Diels. (Apiaceae); and chuanxiong rhizome is the dried rhizome of *L. chuanxiong* Hort. (Apiaceae). All met the standards of the 2020 edition of the *Chinese Pharmacopoeia*. The medicinal materials were randomly combined according to batch number to prepare 15 batches of compound reference samples.

[0100] 2. Methods and Results

[0101] 2.1 Chromatographic conditions

[0102] An Agilent 1260 HPLC system was used with a CAPCELL PAK C18 (MGII) column (4.6 mm × 250 mm, 5 μm). Methanol was used as mobile phase A, acetonitrile as mobile phase B, and 0.2% phosphoric acid solution as mobile phase C. Gradient elution was performed according to the specifications in Table 1. The flow rate was 1.0 mL·min⁻¹. The detection wavelengths were: 0 min – 16 min, 280 nm; 16 min – 30 min, 210 nm; 30 min – 37 min, 230 nm; 37 min – 48 min, 330 nm; 48 min – 51 min, 280 nm; 51 min – 54 min, 403 nm; 54 min – 70 min, 280 nm. The theoretical plate number, calculated based on the gallic acid peak, should be no less than 3000.

[0103] Table 1 Gradient Elution Table of Mobile Phase

[0104]

[0105] 2.2 Solution Preparation

[0106] 2.3.1 Reference solution: Accurately weigh an appropriate amount of gallic acid reference standard, add 25% methanol to prepare a solution containing 50 μg of gallic acid per 1 mL.

[0107] 2.3.2 The test solution is prepared by weighing 56 g of Rehmannia glutinosa (processed with wine), 75 g of Angelica sinensis (processed with wine), 28 g of Paeonia lactiflora (processed with wine), 19 g of Ligusticum chuanxiong, 20 g of Prunus persica (peeled and tip removed), and 19 g of Carthamus tinctorius (processed with wine). The above six herbs are added to 12 times the volume of water, brought to a boil over high heat, then simmered over low heat for 25 minutes. This process is repeated once. The mixture is then filtered, and water is added to a final volume of 1000 mL. The six herbs collected and tested previously are randomly combined according to section “1.2” to prepare 15 batches of compound reference materials. Test solutions are prepared for each batch using the method described above, and these are numbered S1-S15. These compound research samples were provided by the Shanghai Institute of Standardization of Traditional Chinese Medicine.

[0108] 2.4 Feature Mapping

[0109] 2.4.1 Precision Examination

[0110] Take the same batch of test solution (batch number S1), inject it six times consecutively under the chromatographic conditions described in section "2.1", record the chromatograms, and use peak 1 (gallic acid) as the reference peak to calculate the relative retention time and relative peak area of ​​all common peaks. The results show that the RSDs of the relative retention time and relative peak area of ​​each common peak are within the ranges of 0% - 0.20% and 0% - 3.99%, respectively, indicating good instrument precision. The results are shown in Tables 2 and 3.

[0111] Table 2. Precision results of relative retention time of common peaks (n=6)

[0112]

[0113] Table 3. Precision results of common peak relative to retained peak area (n=6)

[0114]

[0115] 2.4.2 Stability Test

[0116] Take the same batch of test solution (batch number S1), and inject it at 0, 2, 6, 12, and 24 h according to the chromatographic conditions in section "2.1", using peak 1 (gallic acid) as the reference peak. Calculate the relative retention time and relative peak area of ​​the 16 common peaks. The results show that the RSDs of the relative retention time and relative peak area of ​​the 16 common peaks are in the range of 0% - 0.35% and 0% - 6.38%, respectively, indicating that the stability of the 16 common peaks of the test solution is good within 24 h at room temperature. The results are shown in Tables 4 and 5.

[0117] Table 4. Results of relative retention time stability of common peaks (n=6)

[0118]

[0119] Table 5. Results of the stability of the common peak relative to the retained peak area

[0120]

[0121] 2.4.3 Repeatability Test

[0122] Six parallel aliquots of the same batch of test solution (batch number S1) were prepared and chromatographically analyzed under the conditions described in section "2.1", using the gallic acid peak (peak 1) as the reference peak. The relative retention times and relative peak areas of the 16 common peaks were calculated. The results showed that the RSD% of the relative retention times and relative peak areas of the 16 common peaks were within the ranges of 0% - 0.41% and 0% - 5.39%, respectively, indicating good repeatability of the method. The results are shown in Tables 6 and 7.

[0123] Table 6. Repeatability results of relative retention time of common peaks

[0124]

[0125] Table 7. Repeatability results of common peak relative to retained peak area

[0126]

[0127] 2.4.4 Establishment and Analysis of Feature Maps

[0128] Take 15 batches of the test solution of the compound (S1-S15) and detect it according to the chromatographic conditions in section "2.1". A total of 16 common peaks were found in the characteristic chromatogram of the test sample. The obtained chromatographic data were imported into the software of the National Pharmacopoeia Commission's "Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine" (2012, 130723 version) for analysis. The relative retention times of peaks 1-16 were calculated with gallic acid peak (peak 1) as reference peak. The relative retention times of all common peaks were set to be within ±5% of the specified values, which were 1.00 (peak 1), 1.08 (peak 2), 1.27 (peak 3), 1.85 (peak 4), 2.14 (peak 5), 2.23 (peak 6), 2.55 (peak 7), 2.64 (peak 8), 2.88 (peak 9), 3.47 (peak 10), 3.78 (peak 11), 4.10 (peak 12), 4.13 (peak 13), 4.23 (peak 14), 4.73 (peak 15), and 4.81 (peak 16). Full-spectrum peak matching was performed to obtain the characteristic spectra of 15 batches of compound test solutions (S1-S15). The results are shown in […]. Figure 1 .

[0129] The results showed that the similarity of the test solutions (S1-S15) of the compound with their reference characteristic chromatograms was greater than 0.9. This indicates that the preparation process of the compound is stable and the differences between different batches are small. A total of 16 common peaks were identified in the whole compound. After comparison with the relevant chromatogram peaks of the reference standard, 13 peaks had the same retention time as the corresponding reference peaks. Peak 1 was identified as gallic acid, peak 3 as rehmannia glycoside D, peak 5 as D-amygynol, peak 6 as chlorogenic acid, peak 7 as hydroxysaffron yellow A, peak 8 as paeoniflorin, peak 9 as paeoniflorin, peak 10 as ferulic acid, peak 11 as verbascoside, peak 13 as ligustilide I, peak 14 as ligustilide H, peak 15 as ligustilide A, and peak 16 as ligustilide. The results are shown in the figure. Figure 2 .

[0130] 2.5 Methodological Investigation of Multi-Indicator Component Content Determination

[0131] 2.5.1 Examination of Linear Relationships

[0132] Accurately weigh appropriate amounts of each reference standard, prepare stock solutions of specific concentrations, and store them in a refrigerator at 4℃ for later use. Detect the solutions under the chromatographic conditions described in section "2.1". Plot a standard curve with the reference standard concentration as the abscissa (x) and the peak area integral as the ordinate (y). Perform linear regression to obtain the regression equation: gallic acid y = 47.766x + 15.32, r = 0.9998, linear range 0.60 - 126.00 μg·mL. -1 Rehmannia glutinosa D y=10.564x-4.2854, r=0.9999, linear range 2.92-203.00 μg·mL -1 amygdalin y = 16.197x + 27.868, r = 0.9999, linear range 2.73 - 364.32 μg·mL -1 Chlorogenic acid y = 54.896x + 2.8473, r = 0.9999, linear range 0.63 - 25.25 μg·mL -1 Hydroxysafflower yellow A y=24.543x-43.61, r=0.9999, linear range 1.51-318.59 μg·mL -1 Paeoniflorin y = 12.706x - 16.479, r = 1.0000, linear range 3.02 - 241.92 μg·mL -1 Paeoniflorin y = 7.0186x + 7.4901, r = 0.9999, linear range 5.55 - 739.68 μg·mL -1Ferulic acid y = 59.03x + 3.9202, r = 1.0000, linear range 0.60 - 158.72 μg·mL -1 Verbascoside y = 28.33x + 3.5346, r = 0.9999, linear range 0.99 - 16.51 μg·mL -1 The linear range for ligustilide I was y = 96.685x + 15.413, r = 0.9999, with a linear range of 0.35 - 70.42 μg·mL. -1 The linear range for ligustilide (Hy = 96.8x + 5.2823, r = 0.9999) is 0.24 - 19.56 μg·mL. -1 The linear range for ligustilide A was y = 16.86x + 3.79533, r = 0.9999, and the linear range was 1.72 - 68.74 μg·mL. -1 The results showed that each component exhibited good linearity within its respective concentration range, as shown in Table 8.

[0133] Table 8 Linear Relationships of Each Component

[0134]

[0135] 2.5.2 Precision Examination

[0136] Accurately pipette the reference solutions of high, medium, and low concentrations as described in section "2.3.1", inject six times repeatedly, and determine the peak area under the chromatographic conditions described in section "2.1". Calculate the intra-day precision results to obtain the RSD of the peak area for each indicator component. For the next three consecutive days, determine the peak area under the chromatographic conditions described in section "2.1" and calculate the inter-day precision results to obtain the RSD of the peak area for each indicator component. The results show that the intra-day and inter-day precision are good, as shown in Tables 9-1, 9-2, 10-1, and 10-2.

[0137] Table 9-1 Results of intraday precision measurement (n=6)

[0138]

[0139] Table 9-2 Results of intraday precision measurement (n=6)

[0140]

[0141] Table 10-1 Results of daytime precision measurement (n=6)

[0142]

[0143] Table 10-2 Results of daytime precision measurement (n=6)

[0144]

[0145] 2.5.3 Repeatability Test

[0146] Six test solutions were prepared in parallel according to the conditions in section "2.3.2" using samples from the same batch (batch number S1). The solutions were then analyzed under the chromatographic conditions in section "2.1". The peak areas of each component were recorded, and the concentrations of each component were calculated using the linear regression equation in section "2.5.1". The RSDs of the concentrations of each index component were obtained. The results showed that the RSDs of the mass fractions of gallic acid, rehmannia glycoside D, amygdalin, chlorogenic acid, hydroxysaffron yellow A, paeoniflorin, paeoniflorin, ferulic acid, verbascoside, ligustrol I, ligustrol H, and ligustrol A were 1.57%, 2.64%, 1.41%, 1.25%, 0.62%, 0.44%, 1.28%, 1.13%, 2.99%, 2.25%, 0.28%, and 1.12%, respectively, indicating that the method had good repeatability. The results are shown in Table 11.

[0147] Table 11 Results of repeatability tests (n=6)

[0148]

[0149] 2.5.4 Stability Assessment

[0150] Take the same sample solution (batch number S1) and prepare 6 parallel sample solutions according to the conditions under section "2.3.2". At room temperature, inject the solutions at 0, 2, 4, 8, 12 and 24 h after preparation under the chromatographic conditions under section "2.1" and record the peak areas of gallic acid, rehmannia glycoside D, amygdalin, chlorogenic acid, hydroxysaffron yellow A, paeoniflorin, paeoniflorin, ferulic acid, verbascoside, ligustrolactone I, ligustrolactone H and ligustrolactone A, and calculate the RSD. The results showed that the RSDs of the peak areas of each component were 0.56%, 1.96%, 1.03%, 1.03%, 0.76%, 1.24%, 1.23%, 1.05%, 1.27%, 1.93%, 0.85%, and 1.48%, respectively. The results indicated that the test solution had good stability within 24 h after preparation. The results are shown in Table 12.

[0151] Table 12 Stability test results (n=6)

[0152]

[0153] 2.5.5 Sample recovery test

[0154] Take the decoction of the compound sample with known content, and add it to the mixed reference solution at 50%, 100%, and 150% of the known content, respectively, three portions of each concentration. Shake well, filter, and collect the filtrate. Accurately pipette 20 μL of each test solution and inject it into the liquid chromatograph. Determine the content according to the chromatographic conditions under section "2.1", record the chromatogram, calculate the content of each component according to the linear regression equation under section "2.5.1", and calculate its recovery rate and RSD. The results showed that the average recoveries of gallic acid, rehmannia glycoside D, amygdalin, chlorogenic acid, hydroxysaffron yellow A, paeoniflorin, paeoniflorin, ferulic acid, verbascoside, ligustilide I, ligustilide H, and ligustilide A were 98.52%, 98.8%, 101.56%, 100.07%, 99.35%, 101.32%, 100.55%, 99.85%, and 99.35%, respectively. The percentages were 0.72%, 101.63%, 103.05%, and 97.12%, with RSD values ​​of 1.83%, 0.91%, 1.98%, 0.98%, 1.58%, 2.24%, 1.14%, 1.07%, 2.57%, 2.37%, 1.49%, and 1.14%, respectively, indicating that the method has good accuracy. The results are shown in Tables 13-1, 13-2, 13-3, and 13-4.

[0155] Table 13-1 Results of the Spiking Recovery Test

[0156]

[0157] Table 13-2 Results of the Spiking Recovery Test

[0158]

[0159] Table 13-3 Results of the spiking recovery test

[0160]

[0161] Table 13-4 Results of the Spiking Recovery Test

[0162]

[0163] 2.5.6 Determination of Sample Content

[0164] Weigh appropriate amounts of 15 batches of compound reference materials accurately, and prepare test solutions according to the test solution preparation method under section "2.3.2". Determine the chromatographic conditions under section "2.1", with each sample measured twice in parallel. Record the peak area of ​​each indicator component. Calculate the contents of gallic acid, rehmannia glycoside D, amygdalin, chlorogenic acid, hydroxysaffron yellow A, paeoniflorin, paeoniflorin, ferulic acid, verbascoside, ligustrolactone I, ligustrolactone H, and ligustrolactone A in the reference materials using the linear regression equation under section "2.5.1". The results are shown in Table 14.

[0165] Table 14 Content of index components in compound reference samples (μg·ml) -1 (n=2)

[0166]

[0167] In Table 14, GA: gallic acid; RD: rehmannia glycoside D; AM: amygdalin; CA: chlorogenic acid; HYA: hydroxysafflower yellow A; AL: paeoniflorin; PA: paeoniflorin; FA: ferulic acid; AC: verbascoside; SI: ligustrazine I; SH: ligustrazine H; SA: ligustrazine A.

[0168] Comparative Example 1

[0169] The test sample was analyzed by liquid chromatography in essentially the same manner as in Example 1, except that the mobile phase used was acetonitrile (mobile phase A) and 0.2% phosphoric acid solution (mobile phase B), and the elution program is shown in Table 15. The detection results of the liquid chromatography are as follows: Figure 3 .

[0170] Table 15 Gradient Elution Table of Mobile Phase

[0171]

[0172] Comparative Example 2

[0173] The test sample was analyzed by liquid chromatography in essentially the same manner as in Example 1, except that the mobile phase used was methanol as mobile phase A and 0.2% phosphoric acid solution as mobile phase B, with the elution program shown in Table 16. The detection results of the liquid chromatography are as follows: Figure 4 .

[0174] Table 16 Gradient Elution Table of Mobile Phase

[0175]

[0176] The chromatographic detection results of Comparative Example 1 ( Figure 3 The chromatographic detection results of Comparative Example 2 and Comparative Example 2 are as follows: Figure 4The results of each chromatographic detection in Example 1 and the obtained characteristic chromatograms ( Figure 1 By comparison, it can be found that when the mobile phase A is entirely composed of acetonitrile or methanol, the baseline stability is poor, the peak signal is weak, the number of peak types is small, and the separation between the characteristic peaks is also poor.

[0177] In summary, this application establishes a characteristic chromatogram of the Taohong Siwu Decoction, a traditional Chinese medicine compound, based on HPLC, and its application in determining the content of multiple components, including gallic acid, rehmannia glycoside D, amygdalin, chlorogenic acid, hydroxysaffron yellow A, paeoniflorin, paeoniflorin, ferulic acid, verbascoside, ligustrolactone I, ligustrolactone H, and ligustrolactone A. The characteristic chromatogram of this application provides an important reference for improving the quality of pharmaceutical preparations and ensuring safe clinical use.

[0178] The characteristic spectrum constructed in this application has a stable baseline, appropriate peak height, good peak shape, and minimal influence from interfering peaks. The similarity of the characteristic spectrum of the 15 batches of samples tested was greater than 0.9, and there was no significant difference in the content of the 12 index components contained therein, indicating that the method is accurate, scientific, and objective.

[0179] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0180] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0181] The embodiments described above merely illustrate several implementation methods of this application and should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Furthermore, it should be understood that after reading the above teachings, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the protection scope of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the protection scope of the appended claims. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A method for constructing a characteristic spectrum of a traditional Chinese medicine compound preparation for treating chronic pelvic inflammatory disease, characterized in that, The traditional Chinese medicine compound preparation for treating chronic pelvic inflammatory disease is Taohong Siwu Decoction. The preparation steps of the Taohong Siwu Decoction include: mixing Rehmannia glutinosa (wine-processed), Angelica sinensis (wine-processed), Paeonia lactiflora (wine-processed), Ligusticum chuanxiong, Prunus persica, and Carthamus tinctorius (wine-processed), adding 11-13 times the amount of water, bringing to a boil over high heat, and then simmering over low heat for 20-30 minutes; the mass ratio of Rehmannia glutinosa (wine-processed), Angelica sinensis (wine-processed), Paeonia lactiflora (wine-processed), Ligusticum chuanxiong, Prunus persica, and Carthamus tinctorius (wine-processed) is (55-57):(74-76):(27-29):(18-20):(19-21):(18-20); The method for constructing the feature map includes the following steps: The tested Taohong Siwu Decoction preparation was dissolved in a solvent to obtain the test solution. The test solution was subjected to liquid chromatography to obtain a characteristic chromatogram of the test sample. The conditions for the liquid chromatography detection include: (1) Methanol is used as mobile phase A, acetonitrile is used as mobile phase B, and 0.1%~0.3% phosphoric acid aqueous solution is used as mobile phase C; (2) Gradient elution is employed, wherein the gradient elution procedure includes: From 0 to 5 min, maintain the volume percentage of mobile phase A at 5%, the volume percentage of mobile phase B at 0%, and the volume percentage of mobile phase C at 95%. From 5 to 15 min, increase the volume percentage of mobile phase A from 5% to 15%, maintain the volume percentage of mobile phase B at 0%, and decrease the volume percentage of mobile phase C from 95% to 85%. From 15 to 18 min, decrease the volume percentage of mobile phase A from 15% to 0%, increase the volume percentage of mobile phase B from 0% to 10%, and increase the volume percentage of mobile phase C from 85% to 90%. From 18 to 45 min, maintain the volume percentage of mobile phase A at 0%, and increase the volume percentage of mobile phase B from 1% to 90%. The volume percentage of mobile phase A increases from 0% to 20%, while the volume percentage of mobile phase C decreases from 90% to 80%; from 45 to 55 minutes, the volume percentage of mobile phase A is maintained at 0%, the volume percentage of mobile phase B increases from 20% to 40%, and the volume percentage of mobile phase C decreases from 80% to 60%; from 55 to 60 minutes, the volume percentage of mobile phase A is maintained at 0%, the volume percentage of mobile phase B increases from 40% to 85%, and the volume percentage of mobile phase C decreases from 60% to 15%; from 60 to 70 minutes, the volume percentage of mobile phase A is maintained at 0%, the volume percentage of mobile phase B increases from 85% to 95%, and the volume percentage of mobile phase C decreases from 15% to 5%. (3) The flow rate is 0.8 mL / min to 1.2 mL / min; (4) 0 min ~ 16 min, detection wavelength is 275 nm ~ 285 nm; 16 min ~ 30 min, detection wavelength is 205 nm ~ 215 nm; 30 min ~ 37 min, detection wavelength is 225 nm ~ 235 nm; 37 min ~ 48 min, detection wavelength is 325 nm ~ 335 nm; 48 min ~ 51 min, detection wavelength is 275 nm ~ 285 nm; 51 min ~ 54 min, detection wavelength is 400 nm ~ 405 nm; 54 min ~ 70 min, detection wavelength is 275 nm ~ 285 nm. (5) The chromatographic column used for the liquid chromatography detection is a C18 column; The characteristic spectrum includes 16 characteristic peaks, of which peak 1 is gallic acid, peak 3 is rehmannia glycoside D, peak 5 is D-amygdalin, peak 6 is chlorogenic acid, peak 7 is hydroxysafflower yellow pigment A, peak 8 is paeoniflorin, peak 9 is paeoniflorin, peak 10 is ferulic acid, peak 11 is verbascoside, peak 13 is ligustilide I, peak 14 is ligustilide H, peak 15 is ligustilide A, and peak 16 is ligustilide.

2. The method for constructing a feature map as described in claim 1, characterized in that, The injection volume for the liquid chromatography detection is 4 μL to 6 μL.

3. The method for constructing a feature map as described in claim 1, characterized in that, The column temperature for the liquid chromatography detection is 28℃~32℃.

4. The method for constructing the feature map as described in any one of claims 1 to 3, characterized in that, It also includes the following steps: A reference solution is provided, which includes gallic acid and a solvent; The reference solution was subjected to liquid chromatography to obtain a characteristic spectrum of the reference. The characteristic spectrum of the reference material was compared with the characteristic spectrum of the test sample, and the characteristic spectrum of the Taohong Siwu Decoction preparation was prepared. The conditions for the liquid chromatography detection are defined as described in any one of claims 1 to 3.

5. The method for constructing the feature map as described in any one of claims 1 to 3, characterized in that, Of the 16 characteristic peaks, with gallic acid peak 1 as the reference peak, the remaining characteristic peaks and their relative retention times are as follows: Peak 2: 1.08±5%; Peak 3: 1.27±5%; Peak 4: 1.85±5%; Peak 5: 2.14±5%; Peak 6: 2.23±5%; Peak 7: 2.55±5%; Peak 8: 2.64±5%; Peak 9: 2.88±5%; Peak 10: 3.47±5%; Peak 11: 3.78±5%; Peak 12: 4.10±5%; Peak 13: 4.13±5%; Peak 14: 4.23±5%; Peak 15: 4.73±5%; Peak 16: 4.81±5%.

6. The application of the method for constructing the characteristic spectrum of the traditional Chinese medicine compound preparation for treating chronic pelvic inflammatory disease according to any one of claims 1 to 5, characterized in that, For use in at least one of the following (1) to (3): (1) Identify the authenticity of the traditional Chinese medicine compound preparation for treating chronic pelvic inflammatory disease; (2) To evaluate the quality of the traditional Chinese medicine compound preparation for treating chronic pelvic inflammatory disease; (3) The content of the indicator components in the traditional Chinese medicine compound preparation for treating chronic pelvic inflammatory disease was determined.

7. The application as described in claim 6, characterized in that, The content of various indicator components in the traditional Chinese medicine compound preparation for treating chronic pelvic inflammatory disease was measured, including: The traditional Chinese medicine compound preparation for treating chronic pelvic inflammatory disease to be tested was dissolved in a solvent to prepare the test solution; Provide a reference solution, including the indicator component and solvent; The test solution and the reference solution were subjected to liquid chromatography for detection, and the content of each indicator component in the test solution was calculated. The conditions for the liquid chromatography detection are defined as described in any one of claims 1 to 3.

8. The application as described in claim 6 or 7, characterized in that, The indicator components include gallic acid, rehmannia glycoside D, amygdalin, chlorogenic acid, hydroxysaffron yellow A, paeoniflorin, paeoniflorin, ferulic acid, verbascoside, ligustrol I, ligustrol H and ligustrol A.

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